Western Blot Analysis of CHAP-amidase

Western blot analysis was conducted as previously reported (Fahimi et al., 2014 (link); Ferdosian et al., 2015 (link); Hosseini et al., 2016 (link)). Briefly, the lysate of CHAP-amidase-expressing cells was run on 10% SDS-PAGE (Bioneer Co., South Korea) and transferred onto nitrocellulose membrane using a semi-dry transfer system. The membrane was blocked overnight in 5% skimmed milk at 4°C and then incubated with primary antibody (anti-His tag monoclonal antibody (mAb) (Abcam Inc., MA, USA), while shaking for 2 h at room temperature (22°C). The membrane was washed three times with PBST (PBS containing 0.05% Tween 20) and incubated with secondary antibody (1:4,000 dilution of HRP-conjugated rabbit anti-mouse IgG antibody) (Abcam Inc., MA, USA) with gentle shaking for 1 h at room temperature. The membrane was washed three times with PBST and the signal was detected by adding DAB (3,3′-diaminobenzidine) as a chromogenic substrate.